DESCRIPTION: There is a clear association between excessive exposure to estrogens and the development of cancer in several tissues including breast and endometrium. The risk factors for women developing these cancers are all associated with longer estrogen exposure; early menses, late menopause, and long term estrogen replacement therapy. The mechanism(s) of estrogen carcinogenesis is not known. The central hypothesis is that the formation of quinoids is an important mechanism of estrogen carcinogenesis. o-Quinones are known metabolites of estrogens. These redox-active compounds have been shown to cause oxidative damage to cellular DNA. Furthermore, these o-quinones can alkylate DNA that may lead to genotoxicity. Our data also suggests that DNA damage may be potentiated by the presence of an estrogen receptor (ER). The specific aims are: 1. What is the predominant mechanism of catechol estrogen-induced DNA damage? Oxidation versus alkylation. Using synthetic oligonucleotides with defined sequences, we will explore the effect of time, concentration, sequence, and redox status on formation of each DNA lesion. Of special interest is the role of the estrogen responsive element since our hypothesis predicts that extensive damage should occur at estrogen sensitive genes. 2. Correlation between DNA damage and estrogen receptor status in cell lines. We will further investigate the role of ERa and/or ERb in mediating catechol estrogen-induced DNA damage and toxicity. The cell lines to be examined include tumorigenic and nontransformed human breast cell lines with no estrogen receptor or ERa or ERb. The comet assay will be employed to examine DNA single strand cleavage and oxidation and alkylation of DNA bases will be studied using LC-MS-MS. 3. Evaluation of the binding affinity of catechol estrogens for ERa and ERb and the functional consequences of ER activation. We propose to evaluate the estrogenic potency of catechol estrogens and correlate these effects with the extent of DNA damage observed in Aims 1 and 2. The relative binding affinity of these compounds to ERa and ERb proteins will be measured using a competitive binding assay. These results will be compared to studies evaluating the ability of the catechol estrogens to transcriptionally activated ER WERE luciferase and ERb /ERE luciferase assays. Finally, we will study the modulation of certain estrogen responsive genes by catechol estrogens in the cell lines described in Aim 2. 4. Role of quinoids in the carcinogenic effects of estrogens. We plan to further explore the relative ability of the catechol estrogens to induce cellular transformation in the non-transformed human breast epithelial cell lines discussed above. The transformed colonies will be implanted into athymic nude mice to establish their ability to induce tumor formation. Finally, to determine whether the parent estrogens are carcinogenic in vivo, ACT rats will be treated with estrogens and the mammary tissue will be analyzed for tumor formation. These data will determine the role of quinoids in the carcinogenic effects of estrogens and provide a basis for the development of estrogen replacement drugs devoid of carcinogenic activity.